Real Time Analysis (RTA3) on NextSeq 1000/2000 Overview

Background: The NextSeq 1000/2000 runs Real-Time Analysis (RTA) 3, an implementation of Real-Time Analysis software on the instrument Compute Engine (CE). RTA3 extracts intensities from images received from the camera, performs base calling, assigns a quality score to base calls, aligns to PhiX, and reports data in InterOp files for viewing in Instrument Control Software and Sequencing Analysis Viewer (SAV).

To optimize processing time, RTA3 stores information in memory. If RTA3 is terminated, processing does not resume and any run data being processed in memory is lost.

RTA3 Inputs: RTA3 requires tile images contained in local system memory for processing. RTA3 receives run information and commands from the control software.

RTA 3 Outputs: Images in each color channel are passed in memory to RTA3 as tiles. From these images, RTA3 outputs a set of quality-scored base call files and filter files. All other outputs are supporting output files.

• Base call files: Each analyzed tile is included in a concantenated base call (\.cbcl) file. Tiles from the same lane and surface are aggregated into 1 \.cbcl file for each lane and surface.

• Filter files: Each tile produces a filter file (\*.filter) that specifies whether a cluster passes filter.

• Cluster location files: Cluster location (\*.locs) files contain the X,Y coordinates for every cluster in a tile. A cluster location file is generated for each run.

Output files are used for downstream analysis in DRAGEN and BaseSpace Sequence Hub.

BCLs are concatenated on the NextSeq 1000/2000 creating one \*.cbcl file per surface for each cycle rather than per tile. Each cycle will have L001_1.cbcl and L001_2.cbcl.

RTA workflow:

1. Registration: Records the location of each cluster on the patterned flow cell.

• Registration aligns an image to the rotated square array of nanowells on the patterned flow cell. Because of the ordered arrangement of nanowells, the X and Y coordinates for each cluster in a tile are predetermined. Cluster positions are written to a cluster location (s.locs) file for each run

2. Intensity extraction: Determines an intensity value for each cluster.

• After registration, intensity extraction calculates an intensity value for each nanowell in a given image.

3. Phasing correction: Corrects the effects of phasing and prephasing.

• During the sequencing reaction, each DNA strand in a cluster extends by one base per cycle. Phasing and prephasing occurs when a strand becomes out of phase with the current incorporation cycle.

  • Phasing occurs when a base falls behind.

  • Prephasing occurs when a base jumps ahead.

• RTA3 corrects the effects of phasing and prephasing, which maximizes the data quality at every cycle throughout the run.

4. Base calling: Determines a base (A, C, G or T) for every cluster.

• NextSeq 1000/2000 Sequencing System uses two-channel sequencing, which requires only two images to encode the data for four DNA bases, one from the green channel and one from the blue channel.

5. Quality scoring: Assigns a quality score to every base call.

• A quality score (Q-score) is a prediction of the probability of an incorrect base call. A higher Q-score implies that a base call is higher quality and more likely to be correct. After the Q-score is determined, results are recorded in base call (\*.cbcl) files

• RTA3 enables faster data processing, reduced file sizes, and simplified Q-score reporting by binning Qscores into a quality table. The three groups in the quality table correspond to marginal (<Q15), medium (~Q20) and high-quality (>Q30) base calls.

Qscore Bin	50, 100, 200 and 300 cycle kits	600 cycle kits
~Q15	12	9
~Q20	26	20
>Q30	34	34

RTA3 Error Handling: RTA3 creates log files and writes them to the Logs folder. Errors are recorded in a text file in \*.log file format.

The following log files are transferred to the final output destination at the completion of processing:* info_00000.log summarizes important run events.

• error_00000.log lists errors that occurred during a run.

• warning_00000.log lists warnings that occurred during a run.

See NextSeq 1000/2000 Sequencing System Product Documentation for more information.

For any feedback or questions regarding this article (Illumina Knowledge Article #2441), contact Illumina Technical Support [e-mail].